Constitutive NF-[unreadable]B activation-persistent activation in the absence of added stimuli-is observed in developing B lymphocytes and in the pathological setting of human cancer. This activity is important for maturation of B cells and survival and chemo/radioresistance of cancer cells. Because NF-[unreadable]B can be activated by a wide variety of distinct mechanisms, constitutive activation may involve complex, deregulated mechanisms in different cancer settings. The long- term goal of this proposal is to increase our understanding of fundamental mechanisms controlling constitutive NF-[unreadable]B activity. There are five NF-[unreadable]B/Rel transcription factors that function as dimers. Two major NF-[unreadable]B complexes are p65:p50 and cRel:p50 heterodimers. Depending on cellular context, one or both of these can be constitutively activated. A unique aspect of this system is that typically activation of these complexes occurs transiently through a feedback loop with its inhibitor, I[unreadable]B[unreadable], which exports NF-[unreadable]B to the cytoplasm to terminate activity. Constitutive NF-[unreadable]B activation thus requires a mechanism to overcome this intrinsic negative feedback regulation and sustain high activity. Based on research during the previous funding period, we hypothesize that p65 and cRel have unique intrinsic mechanisms that establish different constitutive activation threshold involving unique (i) nuclear export mechanisms and (ii) I[unreadable]B[unreadable]-binding affinities. Moreover, these mechanisms are further enhanced in certain cases by (iii) constitutive degradation of newly synthesized I[unreadable]B[unreadable] via a nonclassical pathway that we identified. Specific aims are: 1. To determine the role of nuclear export in regulating constitutive NF-[unreadable]B activation. 2. To reveal the role of I[unreadable]B[unreadable]-binding affinity in specifying constitutive NF-[unreadable]B activation. 3. To further delineate constitutive NF-[unreadable]B activation mechanisms via nonclassical I[unreadable]B[unreadable] degradation. These proposed studies will increase our understanding of fundamental mechanisms controlling specificity and threshold of constitutive activation of different NF-[unreadable]B complexes, which may help identify key mechanisms to induce chemo- and/or radiosensitization in human cancer, regardless of upstream deregulated pathways involved. Finally, they will also generate novel tools that can be used to analyze critical tumor microenvironment components in promoting constitutive NF-[unreadable]B activity in different human malignancies.